The goal of the proposed research is to develop new tools and a systematic approach to capture and experimentally determine the functional consequences (i.e., the phenotype) of human mutations in high-throughput fashion. This work directly addresses the growing challenge of determining mutation functional consequences, especially for the large number of variants of unknown functional significance (VUS's). We will analyze missense mutations in the 15-member Fanconi anemia (or FANC) gene family that plays key roles in genomic stability and the response to DNA damage, as well as in disease risk and therapy. This gene family provides a biologically interesting and clinically relevant target fo this research. Significance: Genome projects are identifying ever larger numbers of mutations of unknown functional significance (VUS's). The proposed research will establish a facile way to rapidly and systematically determine the functional consequences of large numbers (dozens to hundreds) of mutations in parallel in an isogenic, human cell-based assay system using well-defined, clinically relevant endpoints. Approach: We are integrating computational and new experimental tools to determine in a human cell-based assay system the functional phenotype of all missense mutations in the human Fanconi anemia (FANC) genes. Our Aims are: Aim 1: Capture and in silico analysis of all human Fanconi anemia/FANC gene mutations. Aim 2: High-throughput human cell phenotyping of all FANC gene missense mutations. Aim 3: Integrated, data-driven modeling of FANC protein function in response to DNA damage. Innovation: The proposed research has four innovative features: 1. global, ascertainment bias-free capture of all genetic variation in the FANC gene families; 2. systematic mutation functional prediction and rank-scoring using six different prediction algorithms; 3. a new, high-throughput cell-based functional phenotyping assay that is quantitative, and built on clinically relevant cellular endpoints; and 4. the use of mutation phenotyping and molecular data to build an integrated dynamic model of Fanconi protein function in response to DNA damage. Anticipated outcomes: The proposed research will establish a systematic approach for determining the functional phenotype of human mutations including the growing number of VUS's. Results of this work should substantially improve our understanding of Fanconi gene function, while providing a general approach and new tools to enable other human biology and genetic disease research.